Journal of Molecular Modeling

, Volume 18, Issue 5, pp 2031–2042 | Cite as

Transient pockets on XIAP-BIR2: toward the characterization of putative binding sites of small-molecule XIAP inhibitors

  • Susanne Eyrisch
  • Jose L. Medina-Franco
  • Volkhard HelmsEmail author
Original Paper


Protein-protein interactions are abundant in signal transduction pathways and thus of crucial importance in the regulation of apoptosis. However, designing small-molecule inhibitors for these potential drug targets is very challenging as such proteins often lack well-defined binding pockets. An example for such an interaction is the binding of the anti-apoptotic BIR2 domain of XIAP to the pro-apoptotic caspase-3 that results in the survival of damaged cells. Although small-molecule inhibitors of this interaction have been identified, their exact binding sites on XIAP are not known as its crystal structures reveal no suitable pockets. Here, we apply our previously developed protocol for identifying transient binding pockets to XIAP-BIR2. Transient pockets were identified in snapshots taken during four different molecular dynamics simulations that started from the caspase-3:BIR2 complex or from the unbound BIR2 structure and used water or methanol as solvent. Clustering of these pockets revealed that surprisingly many pockets opened in the flexible linker region that is involved in caspase-3 binding. We docked three known inhibitors into these transient pockets and so determined five putative binding sites. In addition, by docking two inactive compounds of the same series, we show that this protocol is also able to distinguish between binders and nonbinders which was not possible when docking to the crystal structures. These findings represent a first step toward the understanding of the binding of small-molecule XIAP-BIR2 inhibitors on a molecular level and further highlight the importance of considering protein flexibility when designing small-molecule protein-protein interaction inhibitors.


Apoptosis Docking Inhibitors Molecular dynamics simulation Pocket detection Protein-protein interaction Transient pockets 



We thank Jan Fuhrmann and Dirk Neumann (Center for Bioinformatics, Saarland University) for making available their BALLPass implementation to us.

Supplementary material

894_2011_1217_MOESM1_ESM.doc (3.9 mb)
ESM 1 (DOC 4033 kb)


  1. 1.
    Wells AL, McClendon CL (2007) Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature 450:1001–1009CrossRefGoogle Scholar
  2. 2.
    Roy N, Deveraux QL, Takahashi R, Salvesen GS, Reed JC (1997) The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J 16:6914–6925CrossRefGoogle Scholar
  3. 3.
    Deveraux QL, Roy N, Stennicke HR, Van Arsdale T, Zhou Q, Srinivasula SM, Alnemri ES, Salvesen GS, Reed JC (1998) IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases. EMBO J 17:2215–2223CrossRefGoogle Scholar
  4. 4.
    Reed JC (2001) The survivin saga goes in vivo. J Clin Invest 108:965–969CrossRefGoogle Scholar
  5. 5.
    Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16Google Scholar
  6. 6.
    Thornberry NA, Lazebnik Y (1998) Caspases: enemies within. Science 281:1312–1316CrossRefGoogle Scholar
  7. 7.
    Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, Scudiero DA, Tudor G, Qui YH, Monks A, Andreeff M, Reed JC (2000) Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias. Clin Cancer Res 6:1796–1803Google Scholar
  8. 8.
    Takahashi R, Deveraux Q, Tamm I, Welsh K, Assa-Munt N, Salvesen GS, Reed JC (1998) A single BIR domain of XIAP sufficient for inhibiting caspases. J Biol Chem 14:7787–7790CrossRefGoogle Scholar
  9. 9.
    Deveraux QL, Leo E, Stennicke HR, Welsh K, Salvesen GS, Reed JC (1999) Cleavage of human inhibitor of apoptosis protein XIAP results in fragments with distinct specificities for caspases. EMBO J 18:5242–5251CrossRefGoogle Scholar
  10. 10.
    Du C, Fang M, Li Y, Li L, Wang X (2000) Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102:33–42CrossRefGoogle Scholar
  11. 11.
    Riedl SJ, Renatus M, Schwarzenbacher R, Zhou Q, Sun C, Fesik SW, Liddington RC, Salvesen GS (2001) Structural basis for the inhibition of caspase-3 by XIAP. Cell 104:791–800CrossRefGoogle Scholar
  12. 12.
    Chai J, Shiozaki E, Srinivasula SM, Wu Q, Datta P, Alnemri ES, Shi Y (2001) Structural basis of caspase-7 inhibition by XIAP. Cell 104:769–780CrossRefGoogle Scholar
  13. 13.
    Shiozaki EN, Chai J, Rigotti DJ, Riedl SJ, Li P, Srinivasula SM, Alnemri ES, Fairman R, Shi Y (2003) Mechanism of XIAP-mediated inhibition of caspase-9. Mol Cell 11:519–527CrossRefGoogle Scholar
  14. 14.
    Liu Z, Sun C, Olejniczak ET, Meadows RP, Betz SF, Oost T, Herrmann J, Wu JC, Fesik SW (2000) Structural basis for binding of Smac/Diablo to the XIAP BIR3 domain. Nature 408:1004–1008CrossRefGoogle Scholar
  15. 15.
    Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y (2000) Structural basis of IAP recognition by Smac/Diablo. Nature 408:1008–1012CrossRefGoogle Scholar
  16. 16.
    Wist AD, Gu L, Riedl SJ, Shi Y, McLendon GL (2007) Structure-activity based study of the Smac-binding pocket within the BIR3 domain of XIAP. Bioorg Med Chem 15:2935–2943CrossRefGoogle Scholar
  17. 17.
    Sun H, Stuckey JA, Nikolovska-Coleska Z, Qin D, Meagher JL, Qiu S, Lu J, Yang CY, Saito NG, Wang S (2008) Structure-based design, synthesis, evaluation, and crystallographic studies of conformationally constrained Smac mimetics as inhibitors of the X-linked inhibitor of apoptosis protein (XIAP). J Med Chem 51:7169–7180CrossRefGoogle Scholar
  18. 18.
    Mastrangelo E, Cossu F, Milani M, Sorrentino G, Lecis D, Delia D, Manzoni L, Drago C, Seneci P, Scolastico C, Rizzo V, Bolognesi M (2008) Targeting the X-linked inhibitor of apoptosis protein through 4-substituted azabicyclo[5.3.0]alkane Smac mimetics. Structure, activity, and recognition principles. J Mol Biol 384:673–689CrossRefGoogle Scholar
  19. 19.
    Nikolovska-Coleska Z, Meagher JL, Jiang S, Yang CY, Qiu S, Roller PP, Stuckey JA, Wang S (2008) Interaction of a cyclic, bivalent Smac mimetic with the x-linked inhibitor of apoptosis protein. Biochemistry 47:9811–9824CrossRefGoogle Scholar
  20. 20.
    Cossu F, Milani M, Mastrangelo E, Vachette P, Servida F, Lecis D, Canevari G, Delia D, Drago C, Rizzo V, Manzoni L, Seneci P, Scolastico C, Bolognesi M (2009) Structural basis for bivalent Smac-mimetics recognition in the IAP protein family. J Mol Biol 392:630–644CrossRefGoogle Scholar
  21. 21.
    Schimmer AD, Welsh K, Pinilla C, Wang Z, Krajewska M, Bonneau MJ, Pedersen IM, Kitada S, Scott FL, Bailly-Maitre B, Glinsky G, Scudiero D, Sausville E, Salvesen G, Nefzi A, Ostresh JM, Houghten RA, Reed JC (2004) Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity. Cancer Cell 5:25–35CrossRefGoogle Scholar
  22. 22.
    Wang Z, Cuddy M, Samuel T, Welsh K, Schimmer A, Hanaii F, Houghten R, Pinilla C, Reed JC (2004) Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors. J Biol Chem 279:48168–48176CrossRefGoogle Scholar
  23. 23.
    Kater AP, Dicker F, Mangiola M, Welsh K, Houghten R, Ostresh J, Nefzi A, Reed JC, Pinilla C, Kipps TJ (2005) Inhibitors of XIAP sensitize CD40-activated chronic lymphocytic leukemia cells to CD95-mediated apoptosis. Blood 106:1742–1748CrossRefGoogle Scholar
  24. 24.
    Scott FL, Denault JB, Riedl SJ, Shin H, Renatus M, Salvesen GS (2005) XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs. EMBO J 24:645–655CrossRefGoogle Scholar
  25. 25.
    Sun C, Cai M, Gunasekera AH, Meadows RP, Wang H, Chen J, Zhang H, Wu W, Xu N, Ng SC, Fesik SW (1999) NMR structure and mutagenesis of the inhibitor-of-apoptosis protein XIAP. Nature 401:818–822CrossRefGoogle Scholar
  26. 26.
    Eyrisch S, Helms V (2007) Transient pockets on protein surfaces involved in protein-protein interaction. J Med Chem 50:3457–3464CrossRefGoogle Scholar
  27. 27.
    Eyrisch S, Helms V (2009) What induces pocket openings on protein surface patches involved in protein-protein interactions? J Comput Aid Mol Des 23:73–86CrossRefGoogle Scholar
  28. 28.
    Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, Olson AJ (1998) Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 19:1639–1662CrossRefGoogle Scholar
  29. 29.
    Berwanger A, Eyrisch S, Schuster I, Helms V, Bernhardt R (2010) Polyamines: naturally occurring small molecule modulators of electrostatic protein-protein interactions. J Inorg Biochem 104:118–125CrossRefGoogle Scholar
  30. 30.
    Lerner MG, Bowman AL, Carlson HA (2007) Incorporating dynamics in E. coli dihydrofolate reductase enhances structure-based drug discovery. J Chem Inf Model 47:2358–2365CrossRefGoogle Scholar
  31. 31.
    Bowman AL, Lerner MG, Carlson HA (2007) Protein flexibility and species specificity in structure-based drug discovery: dihydrofolate reductase as a test system. J Am Chem Soc 129:3634–3640CrossRefGoogle Scholar
  32. 32.
    Bogan AA, Thorn KS (1998) Anatomy of hot spots in protein interfaces. J Mol Biol 280:1CrossRefGoogle Scholar
  33. 33.
    Clackson T, Wells J (1995) A hot-spot of binding energy in a hormone-receptor interface. Science 267:383–386CrossRefGoogle Scholar
  34. 34.
    Moreira IS, Fernandes PA, Ramos MJ (2007) Hot spots-a review of the protein-protein interface determinant amino-acid residues. Proteins 68:803–812CrossRefGoogle Scholar
  35. 35.
    Halperin I, Wolfson H, Nussinov R (2004) Protein-protein interactions: coupling of structurally conserved residues and of hot spots across interfaces. Implications for docking. Structure 12:1027–1038CrossRefGoogle Scholar
  36. 36.
    Keskin O, Ma B, Nussinov R (2005) Hot regions in protein-protein interactions: the organization and contribution of structurally conserved hot spot residues. J Mol Biol 345:1281–1294CrossRefGoogle Scholar
  37. 37.
    Kortemme T, Baker D (2002) A simple physical model for binding energy hot spots in protein-protein complexes. Proc Natl Acad Sci USA 99:14116–14121CrossRefGoogle Scholar
  38. 38.
    Guerois R, Nielsen J, Serrano L (2002) Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J Mol Biol 320:369–387CrossRefGoogle Scholar
  39. 39.
    Tuncbag N, Gursoy A, Keskin O (2009) Identification of computational hot spots in protein interfaces: combining solvent accessibility and inter-residue potentials improves the accuracy. Bioinformatics 25:1513–1520CrossRefGoogle Scholar
  40. 40.
    Bylaska EJ, de Jong WA, Kowalski K, Straatsma TP, Valiev M et al (2006) NWChem, a computational chemistry package for parallel computers, version 5.0, Pacific Northwest National Laboratory, Richland, Washington 99352-0999, USAGoogle Scholar
  41. 41.
    Becke AD (1997) Density-functional thermochemistry. V. Systematic optimization of exchange-correlation functionals. J Chem Phys 107:8554CrossRefGoogle Scholar
  42. 42.
    Bayly CI, Cieplak P, Cornell W, Kollman PA (1993) A well-behaved electrostatic potential based method using charge restraints for determining atom-centered charges: the RESP model. J Phys Chem 97:10269–10280CrossRefGoogle Scholar
  43. 43.
    Jorgensen WL, Maxwell DS, Tirado-Rives J (1996) Development and testing of the opls all-atom force field on conformational energetics and properties of organic liquids. J Am Chem Soc 118:11225–11236CrossRefGoogle Scholar
  44. 44.
    Merz KM (1991) Carbon dioxide binding to human carbonic anhydrase ii. J Am Chem Soc 113:406–411CrossRefGoogle Scholar
  45. 45.
    Lindahl E, Hess B, van der Spoel D (2001) GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model 7:306–317Google Scholar
  46. 46.
    Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935CrossRefGoogle Scholar
  47. 47.
    Darden T, York D, Pedersen L (1993) Particle mesh ewald: an n log(n) method for ewald sums in large systems. J Chem Phys 98:10089–10092CrossRefGoogle Scholar
  48. 48.
    Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690CrossRefGoogle Scholar
  49. 49.
    Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472CrossRefGoogle Scholar
  50. 50.
    Brady GP Jr, Stouten PF (2000) Fast prediction and visualization of protein binding pockets with pass. J Comput Aided Mol Des 14:383–401CrossRefGoogle Scholar
  51. 51.
    Gasteiger J, Marsili M (1980) Iterative partial equilibration of orbital electronegativity – a rapid access to atomic charges. Tetrahedron 36:3219–3228CrossRefGoogle Scholar
  52. 52.
    Sanner MF (1999) Python: a programming language for software integration and development. J Mol Graph Model 17:57–61Google Scholar
  53. 53.
    Solis FJ, Wets JB (1981) Minimization by random search techniques. Math Oper Res 6:19–30CrossRefGoogle Scholar
  54. 54.
    Huey R, Morris GM, Olson AJ, Goodsell DS (2007) A semiempirical free energy force field with charge-based desolvation. J Comput Chem 28:1145–1152CrossRefGoogle Scholar
  55. 55.
    Boehr DD, Wright PE (2008) How do proteins interact? Science 320:1429–1430CrossRefGoogle Scholar
  56. 56.
    Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791CrossRefGoogle Scholar
  57. 57.
    Obiol-Pardo C, Granadino-Roldan JM, Rubio-Martinez J (2008) Protein-protein recognition as a first step towards the inhibition of XIAP and Survivin anti-apoptotic proteins. J Mol Recognit 21:190–204CrossRefGoogle Scholar
  58. 58.
    Pang YP, Xu K, El Yazal J, Prendergast PG (2000) Successful molecular dynamics simulation of the zinc-bound farnesyltransferase using the cationic dummy atom approach. Protein Sci 9:1857–1865Google Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Susanne Eyrisch
    • 1
    • 2
  • Jose L. Medina-Franco
    • 3
  • Volkhard Helms
    • 1
    Email author
  1. 1.Center for BioinformaticsSaarbrueckenGermany
  2. 2.Priaxon AGMünchenGermany
  3. 3.Torrey Pines Institute for Molecular StudiesPort St. LucieUSA

Personalised recommendations